Histamine synthesis by purified intact rat peritoneal mast cells, as measured by formation of Beta-3H-L-histidine or release of 14C02 from 14C-carboxyl-labeled histidine, was 10 to 30 times greater than that of disrupted cells or soluble extracts of these cells. Loss of activity was evident whether cells were disrupted by sonification, freezing and thawing, or lysis both in the absence and presence of agents known to preserve enzyme activity. Studies with decarboxylase inhibitors indicated that a specific histidine decarboxylase was responsible for histamine formation in both the intact cells and cell extracts. In the presence of subsaturating concentrations of histidine, various histidine analogs and glutamine inhibit histidine uptake and histamine formation in intact mass cells but did not inhibit synthesis in cell extracts. These data indicate that, at physiological concentrations of histidine, blockade of histidine transport may limit histamine synthesis in the intact cell and that measurement of histidine decarboxylase activity in tissue homogenates or cell extracts may not reflect the rate of histamine synthesis in vivo.